Chemical modification of proteins to improve biocompatibility and bioactivity

ABSTRACT

The present invention broadly relates to chemical modification of biologically active proteins or analogs thereof. More specifically, the present invention describes novel methods for site-specific chemical modification of various proteins, and resultant compositions having improved biocompatibility and bioactivity.

FIELD OF THE INVENTION

The present invention broadly relates to chemical modification ofbiologically active proteins or analogs thereof (the term "protein" asused herein is synonymous with "polypeptide" or "peptide" unlessotherwise indicated). More specifically, the present invention describesnovel methods for site-specific chemical modifications of variousproteins, and resultant compositions.

BACKGROUND OF THE INVENTION

Due to recent advances in genetic and cell engineering technologies,proteins known to exhibit various pharmacological actions in vivo arecapable of production in large amounts for pharmaceutical applications.Such proteins include erythropoietin (EPO), granulocytecolony-stimulating factor (G-CSF), interferons (alpha, beta, gamma,consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF),kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocytegrowth differentiation factor (MGDF), osteoprotegerin (OPG), glial cellline derived neurotrophic factor (GDNF) and obesity protein (OBprotein). OB protein may also be referred to herein as leptin.

Leptin is active in vivo in both ob/ob mutant mice (mice obese due to adefect in the production of the OB gene product) as well as in normal,wild type mice. The biological activity manifests itself in, among otherthings, weight loss. See generally, Barinaga, "Obese" Protein SlimsMice, Science 269: 475-476 (1995) and Friedman, "The Alphabet of WeightControl," Nature 385: 119-120 (1997). It is known, for instance, that inob/ob mutant mice, administration of leptin results in a decrease inserum insulin levels, and serum glucose levels. It is also known thatadministration of leptin results in a decrease in body fat. This wasobserved in both ob/ob mutant mice, as well as non-obese normal mice.Pelleymounter et al., Science 269: 540-543 (1995); Halaas et al.,Science 269: 543-546 (1995). See also, Campfield et al., Science 269:546-549 (1995) (Peripheral and central administration of microgram dosesof leptin reduced food intake and body weight of ob/ob and diet-inducedobese mice but not in db/db obese mice.) In none of these reports havetoxicities been observed, even at the highest doses.

Preliminary leptin induced weight loss experiments in animal modelspredict the need for a high concentration leptin formulation withchronic administration to effectively treat human obesity. Dosages inthe milligram protein per kilogram body weight range, such as 0.5 or 1.0mg/kg/day or below, are desirable for injection of therapeuticallyeffective amounts into larger mammals, such as humans. An increase inprotein concentration is thus necessary to avoid injection of largevolumes, which can be uncomfortable or possibly painful to the patient.

Unfortunately, for preparation of a pharmaceutical composition forinjection in humans, it has been observed that the leptin amino acidsequence is insoluble at physiologic pH at relatively highconcentrations, such as above about 2 mg active protein/milliliter ofliquid. The poor solubility of leptin under physiological conditionsappears to contribute to the formation of leptin precipitates at theinjection site in a concentration dependent manner when high dosages areadministered in a low pH formulation. Associated with the observedleptin precipitates is an inflammatory response at the injection sitewhich includes a mixed cell infiltrate characterized by the presence ofeosinophils, macrophages and giant cells.

To date, there have been no reports of stable preparations of human OBprotein at concentrations of at least about 2 mg/ml at physiologic pH,and further, no reports of stable concentrations of active human OBprotein at least about 50 mg/ml or above. The development of leptinforms which would allow for high dosage without the aforementionedproblems would be of great benefit. It is therefore one object of thepresent invention to provide improved forms of leptin by way ofsite-specific chemical modification of the protein.

There are several methods of chemical modification of useful therapeuticproteins which have been reported. One such method, succinylation,involves the conjugation of one or more succinyl moieties to abiologically active protein. Classic approaches to succinylationtraditionally employ alkaline reaction conditions with very largeexcesses of succinic anhydride. The resultant succinyl-proteinconjugates are typically modified at multiple sites, often show alteredtertiary and quaternary structures, and occasionally are inactivated.The properties of various succinylated proteins are described inHolcenberg et al., J. Biol. Chem, 250:4165-4170 (1975), and WO 88/01511(and references cited therein), published Mar. 10, 1988. Importantly,none of the cited references describe methods wherein the biologicallyactive protein is monosuccinylated exclusively at the N-terminus of theprotein, and wherein the resultant composition exhibits improvedsolubility and improved injection site toxicity's.

Diethylenetriaminepentaacetic acid anhydride (DTPA) andethylenediaminetetraacetic acid dianhydride (hereinafter referred to asEDTA²) have classically been used to introduce metal chelation sitesinto proteins for the purpose of radiolabeling. Similar tosuccinylation, modification with DTPA and/or EDTA² typically occurs atmultiple sites throughout the molecule and changes the charge andisoelectric point of the modified protein. To date, there have been noreports of DTPA- and/or EDTA² -protein monomers and dimers which exhibitimproved solubility and improved injection site toxicity's.

SUMMARY OF THE INVENTION

The present invention relates to substantially homogenous preparationsof chemically modified proteins, e.g. leptin, and methods therefor.Unexpectedly, site-specific chemical modification of leptin demonstratedadvantages in bioavailibility and biocompatibility which are not seen inother leptin species. Importantly, the methods described herein arebroadly applicable to other proteins (or analogs thereof), as well asleptin. Thus, as described below in more detail, the present inventionhas a number of aspects relating to chemically modifying proteins (oranalogs thereof) as well as specific modifications of specific proteins.

In one aspect, the present invention relates to a substantiallyhomogenous preparation of mono-succinylated leptin (or analog thereof)and related methods. Importantly, the method described results in a highyield of monosuccinylated protein which is modified exclusively at theN-terminus, thereby providing processing advantages as compared to otherspecies. And, despite the modest N-terminal modification, themonosubstituted succinyl-leptin unexpectedly demonstrated: 1) asubstantial improvement in solubility; 2) preservation of secondarystructure, in vitro receptor binding activity and in vivo bioefficacy;and 3) amelioration of the severe injection site reactions observed withadministration of high concentrations of unmodified leptin.

In another aspect, the present invention relates to substantiallyhomogenous preparations of DTPA-leptin monomers and dimers and relatedmethods. When reacted with leptin at neutral pH and a low stoichiometricexcess of DTPA:protein, this reagent unexpectedly forms a singlecrosslink between the N-termini of two leptin molecules in high yield.When the monosubstituted DTPA-leptin monomer and dimer are isolated,both show substantially increased solubility's relative to theunmodified protein. Both forms also demonstrate preservation of in vitroreceptor binding activity and in vivo bioefficacy. Significantly, thedimeric form of monosubstituted DTPA-leptin did not precipitate wheninjected at high concentration in PBS and demonstrated strongimprovement in the injection site reactions over those observed with theunmodified leptin.

In yet another aspect, the present invention relates to substantiallyhomogenous preparations of EDTA dianhydride (EDTA²)-leptin monomers anddimers and related methods. Similar to DTPA in structure, EDTA²crosslinks leptin efficiently through the N-terminus when allowed toreact at neutral pH in a substoichiometric excess. The isolated EDTA²-leptin dimer demonstrates dramatically enhanced solubility relative tounmodified leptin and maintains full in vitro receptor binding activityand in vivo bioactivity. Furthermore, the EDTA² -leptin conjugate didnot precipitate at the injection site when dosed at high concentrationin PBS and demonstrated substantial improvement in the adverse injectionsite reactions observed with the unmodified leptin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram of a anion exchange chromatography separationof succinylated leptin. Absorbance at 280 nm is plotted vs. elutionvolume in mL. The monosuccinylated leptin peak is marked by (*).

FIG. 2 is a pH 3-7 IEF-PAGE gel depicting unmodified leptin (lane 2),succinylated leptin (lane 3), DTPA modified leptin dimer (lane 4) andEDTA² modified leptin dimer (lane 5). Lanes 1 and 6 are isoelectricpoint markers.

FIG. 3 is a chromatogram of a size exclusion chromatography separationof DTPA crosslinked leptin monomer and dimer. Absorbance at 280 nm isplotted vs. elution volume in mL. The dimeric form of monosubstitutedDTPA-leptin is marked by (*)

FIG. 4 is a 4-20% SDS-PAGE gel depicting unmodified leptin (lane 2),succinylated leptin (lane 3), DTPA modified leptin dimer (lane 4) andEDTA² modified leptin dimer (lane 5). Lanes 1 and 6 are molecular weightmarkers.

FIG. 5 is a chromatogram of a size exclusion chromatography separationof EDTA² crosslinked leptin monomer and dimer. Absorbance at 280 nm isplotted vs. elution volume in mL. The dimeric form of monosubstitutedEDTA² -leptin is marked by (*).

FIG. 6 is a reverse phase HPLC chromatogram of Lys-C digests showingretention time shifts resulting from chemical modifications of theN-terminal peptide (M1-K6) by succinic anhydride.

FIG. 7 is a reverse phase HPLC chromatogram of Lys-C digests showingretention time shifts resulting from chemical modifications of theN-terminal peptide (M1-K6) by DTPA.

FIG. 8 is a reverse phase HPLC chromatogram of Lys-C digests showingretention time shifts resulting from chemical modifications of theN-terminal peptide (M1-K6) by EDTA².

FIG. 9 depicts Far-UV CD spectra of unmodified native leptin andmonosuccinylated leptin. Both samples are at 0.25 mg/mL in phosphatebuffered saline at ambient temperature.

FIG. 10 is a graph depicting in vitro receptor binding of unmodifiedleptin (-♦-), succinylated-leptin (-▪-), DTPA-leptin dimer (-▴-) orEDTA² -leptin dimer (--) by displacement of radiolabeled human leptinfrom immobilized human leptin receptor. Ligand concentration (ng/mL) isplotted versus % ligand bound.

FIG. 11 is a graph depicting weight loss in mice that had been treatedwith either unmodified leptin (-♦-), succinylated-leptin (-▪-),DTPA-leptin dimer (-▴-) or DTPA-leptin monomer (-x-). Mice were doseddaily at 10 mg/kg delivered at 2 mg/mL in PBS. Time (days) is plottedversus % weight loss.

FIG. 12 is a graph depicting weight loss in mice that had been treatedwith either 20 mg/mL unmodified leptin (-▴-), 2 mg/mL unmodified leptin(-♦-), 20 mg/mL EDTA² -leptin dimer (-▪-) or 2 mg/mL EDTA² -leptin dimer(-x-). Mice were dosed daily at 100 mg/kg delivered at 20 mg/mL or 10mg/kg delivered at 2 mg/mL in PBS (unmodified leptin dosed at 100 mg/kgand 20 mg/mL was formulated in pH 4.0, acetate buffer due to its poorsolubility in PBS). Time (days) is plotted versus % weight loss.

DETAILED DESCRIPTION

The present invention relates to substantially homogenous preparationsof chemically modified proteins, and methods therefor. "Substantiallyhomogenous" as used herein means that the only chemically modifiedproteins observed are those having one "modifier" (e.g., DTPA, EDTA²,succinyl) moiety. The preparation may contain unreacted (i.e., lackingmodifier moiety) protein. As ascertained by peptide mapping andN-terminal sequencing, one example below provides for a preparationwhich is at least 90% modified protein, and at most 10% unmodifiedprotein. Preferably, the chemically modified material is at least 95% ofthe preparation (as in the working example below) and most preferably,the chemically modified material is 99% of the preparation or more. Thechemically modified material has biological activity. The present"substantially homogenous" monosuccinylated leptin, DTPA-leptin, andEDTA² -leptin preparations provided herein are those which arehomogenous enough to display the advantages of a homogenous preparation,e.g., ease in clinical application in predictability of lot to lotpharmacokinetics.

As used herein, biologically active agents refers to recombinant ornaturally occurring proteins, whether human or animal, useful forprophylactic, therapeutic or diagnostic application. The biologicallyactive agent can be natural, synthetic, semi-synthetic or derivativesthereof. In addition, biologically active agents of the presentinvention can be perceptible. A wide range of biologically active agentsare contemplated. These include but are not limited to hormones,cytokines, hematopoietic factors, growth factors, antiobesity factors,trophic factors, anti-inflammatory factors, and enzymes (see also U.S.Pat. No. 4,695,463 for additional examples of useful biologically activeagents). One skilled in the art will readily be able to adapt a desiredbiologically active agent to the compositions of present invention.

Such proteins would include but are not limited to interferons (see,U.S. Pat. Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 herebyincorporated by reference including drawings), interleukins (see, U.S.Pat. No. 5,075,222, hereby incorporated by reference includingdrawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,5,618,698 5,547,933, and 5,621,080 hereby incorporated by referenceincluding drawings), granulocyte-colony stimulating factors (see, U.S.Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCTPublication No. 94/17185, hereby incorporated by reference includingdrawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and95/17206, hereby incorporated by reference including drawings), andleptin (OB protein) (see PCT publication Nos. 96/40912, 96/05309,97/00128, 97/01010 and 97/06816 hereby incorporated by referenceincluding figures). PCT publication No. WO 96/05309, published Feb. 22,1996, entitled, "Modulators of Body Weight, Corresponding Nucleic Acidsand Proteins, and Diagnostic and Therapeutic Uses Thereof" fully setsforth OB protein and related compositions and methods, and is hereinincorporated by reference. An amino acid sequence for human OB proteinis set forth at WO 96/05309 Seq. ID Nos. 4 and 6 (at pages 172 and 174of that publication), and the first amino acid residue of the matureprotein is at position 22 and is a valine residue. The mature protein is146 residues (or 145 if the glutamine at position 49 is absent, Seq. IDNo. 4).

In addition, biologically active agents can also include but are notlimited to insulin, gastrin, prolactin, adrenocorticotropic hormone(ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH),follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG),motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12),tumor necrosis factor (TNF), tumor necrosis factor-binding protein(TNF-bp), brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblastgrowth factors (FGF), neurotrophic growth factor (NGF), bone growthfactors such as osteoprotegerin (OPG), insulin-like growth factors(IGFs), macrophage colony stimulating factor (M-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), megakaryocyte derivedgrowth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin,platelet-derived growth factor (PGDF), colony simulating growth factors(CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD),tissue plasminogen activator (TPA), urokinase, streptokinase andkallikrein. The term proteins, as used herein, includes peptides,polypeptides, consensus molecules, analogs, derivatives or combinationsthereof.

In general, comprehended by the invention are pharmaceuticalcompositions comprising effective amounts of chemically modifiedprotein, or derivative products, together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers needed for administration. (See PCT 97/01331 herebyincorporated by reference.) The optimal pharmaceutical formulation for adesired biologically active agent will be determined by one skilled inthe art depending upon the route of administration and desired dosage.Exemplary pharmaceutical compositions are disclosed in Remington'sPharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, PA.,pgs. 1435-1712 (1990)). The pharmaceutical compositions of the presentinvention may be administered by oral and non-oral preparations (e.g.,intramuscular, subcutaneous, transdermal, visceral, IV (intravenous), IP(intraperitoneal), intraarticular, placement in the ear, ICV(intracerebralventricular), IP (intraperitoneal), intraarterial,intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal,rectal, and uterine-transmucosal preparations).

Therapeutic uses of the compositions of the present invention depend onthe biologically active agent used. One skilled in the art will readilybe able to adapt a desired biologically active agent to the presentinvention for its intended therapeutic uses. Therapeutic uses for suchagents are set forth in greater detail in the following publicationshereby incorporated by reference including drawings. Therapeutic usesinclude but are not limited to uses for proteins like interferons (see,U.S. Pat. Nos. 5,372,808, 5,541,293, hereby incorporated by referenceincluding drawings), interleukins (see, U.S. Pat. No. 5,075,222, herebyincorporated by reference including drawings), erythropoietins (see,U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080hereby incorporated by reference including drawings), granulocyte-colonystimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476,5,582,823, 4,810,643 and PCT Publication No. 94/17185, herebyincorporated by reference including drawings), stem cell factor (PCTPublication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated byreference including drawings), and the OB protein (see PCT publicationNos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 herebyincorporated by reference including figures). In addition, the presentcompositions may also be used for manufacture of one or more medicamentsfor treatment or amelioration of the conditions the biologically activeagent is intended to treat.

The principal embodiment of the method for making the substantiallyhomogenous preparation of monosuccinylated protein comprises: (a)reacting a protein with 3-7 fold molar excess of succinic anhydride; (b)stirring the reaction mixture 2-16 hours at 4° C.; (c) dialyzing saidmixture against 20 mM Tris-HCl, pH 7.2; and (d) isolating saidmonosuccinylated protein. Optionally, the method can comprise, justafter step (b), the steps of: adding solid hydroxylamine to said mixturewhile maintaining the pH above 6.5 until said hydroxylamine iscompletely dissolved, followed by elevating the pH to 8.5 using 5N NaOH,followed by stirring said mixture another 1-2 hours at 4° C. The generalprocess is shown schematically in Example 1.

The principal embodiment of the method for making the substantiallyhomogenous preparation of DTPA-protein comprises: (a) reacting a proteinwith 1-5 fold molar excess of DTPA; (b) stirring the reaction mixture2-16 hours at 4° C.; (c) dialyzing said mixture against 20 mM Tris-HCl,pH 7.2; and (d) isolating said DTPA-protein. The general process isshown schematically in Example 1.

The principal embodiment of the method for making the substantiallyhomogenous preparation of EDTA² -protein comprises: (a) reacting aprotein with 0.5-5 fold molar excess of EDTA² ; (b) stirring thereaction mixture 2-16 hours at 4° C.; (c) filtering said reactionmixture; (d) concentrating said reaction mixture; and (e) isolating saidEDTA² -protein. The general process is shown schematically in Example 1.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.Example 1 describes the preparation of monosuccinylated leptin,monosubstituted DTPA-leptin monomers and dimers, and EDTA² -leptinmonomers and dimers. Example 2 describes the physiochemicalcharacterization of the modified leptin species prepared in Example 1.Example 3 describes the receptor binding studies performed on themodified leptin species prepared in Example 1. Example 4 describes thesolubility testing performed on the modified leptin species prepared inExample 1. Example 5 describes the in vivo bioactivity studies performedon the modified leptin species prepared in Example 1. Example 6describes the injection site evaluation performed on the modified leptinspecies prepared in Example 1.

EXAMPLE 1

This example describes the preparation of monosuccinylated leptin,monosubstituted DTPA-leptin monomers and dimers, and EDTA² -leptinmonomers and dimers.

1. Monosuccinylated leptin

The protein succinylation method of the present invention can begenerally depicted as follows: ##STR1##

Recombinant human-methionyl-leptin (rhu-met-leptin) protein (prepared asdescribed in Materials and Methods, infra) at 2-3 mg/mL in 20 mM NaHPO₄,pH 7.0, was reacted with 3-7 fold molar excess of solid succinicanhydride (Sigma Chemical, St. Louis, Mo.), with a 5 fold molar excesspreferred, and the reaction stirred 2-16 hours at 4° C. Solidhydroxylamine (Sigma Chemical, St. Louis, Mo.) is then added to thereaction while maintaining the pH above 6.5. After the hydroxylamine hasdissolved completely the pH is elevated to 8.5 using 5N NaOH and thereaction allowed to stir another 1-2 hours at 4° C. (the hydroxylaminestep may be omitted with a small decrease in yield). Finally, thereaction is dialyzed against 20 mM Tris-HCl, pH 7.2.

The monosuccinylated rhu-met-leptin is isolated by anion exchangechromatography with a High Performance Sepharose Q column (Pharmacia,Piscataway, N.J.) in 20 mM Tris, pH 7.2, with a 0-0.5M NaCl gradient(see FIG. 1). The product is recognized in the eluant by an isoelectricshift of -0.7 pI units observed with isoelectric focusing (IEF) PAGEusing a 5% polyacrylamide, pH 3-7 gel (Novex, Inc., San Diego, Calif.)(FIG. 2). Final recovery of monosuccinylated rhu-met-leptin is typically45-47%.

2. Monosubstituted DTPA-leptin monomers and dimers

The DTPA modification method of the present invention can be generallydepicted as follows: ##STR2##

Recombinant human-methionyl-leptin (rhu-met-leptin) protein (prepared asdescribed in Materials and Methods, infra) at 2-3 mg/mL in 20 mM NaHPO₄,pH 7.0, was reacted with a 1-5 fold molar excess of solid DTPA (SigmaChemical, St. Louis, Mo.), with 2-3 fold molar excess preferred, and thereaction stirred 2-16 hours at 4° C. Finally, the reaction is dialyzedagainst 20 mM Tris-HCl, pH 7.2. The DTPA modified rhu-met-leptin isisolated by anion exchange chromatography with a High PerformanceSepharose Q column (Pharmacia, Piscataway, N.J.) in 20 mM Tris, pH 7.2,with a 0-0.5M NaCl gradient. Alternatively, monomeric and dimeric formsof monosubstituted DTPA-rhu-met-leptin or rhu-met-leptin are separatedby size exclusion chromatography on a Sephacryl 100 column (Pharmacia,Piscataway, N.J.) in PBS (Life Technologies, Grand Island, N.Y.) (seeFIG. 3). The products are recognized in the eluant by an isoelectricshift observed with the monomeric DTPA-leptin by isoelectric focusing(IEF) PACE using a 5% polyacrylamide, pH 3-7 gel (Novex, Inc., SanDiego, Calif.) (FIG. 2) or the mass increase of a crosslinked dimerobserved with SDS-PAGE using a 4-20% polyacrylamide gel (Novex, Inc.,San Diego, Calif.) (see FIG. 4). Final recovery of DTPA-rhu-met-leptindimer is approximately 30%.

3. Monosubstituted EDTA² leptin monomers and dimers

The EDTA² modification method of the present invention can be generallydepicted as follows: ##STR3##

Recombinant human-methionyl-leptin (rhu-met-leptin) protein (prepared asdescribed in Materials and Methods, infra) at 2-3 mg/mL in 20 mM NaHPO₄,pH 7.0, was reacted with a 0.5-5 fold molar excess of EDTA² (AldrichChemical Co., Milwaukee, Wis.) either as a solid or dissolved in DMSO,with 0.75 fold molar excess EDTA² in DMSO preferred, and the reactionstirred 2-16 hours at 4° C.

The reaction is then filtered through a 0.45 micron filter (Nalgene),concentrated by stirred cell over 10 kDa molecular weight cutoffmembrane to ˜20 mg/mL and the monomeric and dimeric forms ofmonosubstituted EDTA² -rhu-met-leptin then separated by size exclusionchromatography on a Sephacryl 100 column (Pharmacia, Piscataway, N.J.)equilibrated in PBS (see FIG. 5). Alternatively, the reaction may bepurified by hydrophobic interaction chromatography using a HighPerformance Phenyl-Sepharose column (Pharmacia, Piscataway, N.J.) elutedwith a 0.8-0M ammonium sulfate gradient in 20 mM NaHPO₄, pH 7.0. Theproducts are recognized in the eluant by an isoelectric shift observedwith the monomeric EDTA² -rhu-met-leptin by isoelectric focusing (IEF)PAGE using a 5% polyacrylamide, pH 3-7 gel (Novex, Inc., San Diego,Calif.) (FIG. 2) or the mass increase of a crosslinked dimer observedwith SDS-PAGE using a 4-20% polyacrylamide gel (Novex, Inc., San Diego,Calif.) (FIG. 4). Final recovery of EDTA² -rhu-met-leptin dimer exceeds50%.

EXAMPLE 2

This example describes the physiochemical characterization of the leptinconjugates prepared in Example 1. Modification of succinyl-leptin,DTPA-leptin monomers and dimers, and EDTA² -leptin monomers and dimerswas evaluated by a combination of peptide mapping of Lys-C digests onreverse phase HPLC, MALDI-TOF mass spectrometry and peptide sequencing.

Lys-C digests of unmodified leptin and the various modified leptins wereperformed by reaction of 100 μg of protein with 4 μg of endoproteinaseLys-C (Boehringer Mannheim) in 50 mM Tris-HCl, pH 8.5 (200 μl) for fourhours at room temperature. Peptide maps of the various samples weregenerated by reverse phase HPLC on a 4.6×250 mm, 5μ C4 column (VydaK,Hesperia, Calif.) equilibrated in 0.1% triflouroacetic acid (TFA) withelution over a 0-90% acetonitrile gradient (see FIGS. 6-8). As evidencedby the plots depicted in FIGS. 6-8, only the N-terminal peptide (M1-K6)shows any change in retention time as a result of chemical modification.This result indicates that lysine at position 6 is unmodified andaccessible to Lys-C digestion and suggests that the chemicalmodification occurs at the α-amine of the N-terminus. N-terminalmodification is further supported by efforts at N-terminal sequencingwhich indicate that the N-terminus is blocked (data not shown).

Mass determinations for succinyl-leptin and DTPA- and EDTA² -leptindimers were made on a Kompact Maldi IV (Kratos, Ramsey, N.J.) using a 12pmol sample in a sinapinic acid matrix. Each conjugate indicates asingle chemical modification per molecule.

                  TABLE 1    ______________________________________               Expected Mass                           Linker Mass                                     Measured Mass    Conjugate  (Da)        (Da)      (Da)    ______________________________________    Unmod. leptin               16,157      0         16,156    Succinyl-leptin               16,258      101       16,254    DTPA-leptin dimer               32,671      357       32,705    EDTA.sup.2 -leptin dimer               32,570      256       32,509    ______________________________________

In addition to the analysis above, the effects on the secondarystructure of the succinyl-leptin was evaluated using circular dichroismspectroscopy. Far-UV circular dichroism spectra of unmodified andsuccinylated leptin in phosphate buffered saline were collected using a0.05 cm cell in a Jasco J-710 circular dichroism spectrophotometer(Jasco, Tokyo, Japan). The spectra are depicted in FIG. 9 anddemonstrate that the secondary structure of succinylated-leptin ispreserved.

In sum, the Example 2 data confirms the modification of succinyl-leptin,DTPA-leptin monomers and dimers, and EDTA² -leptin monomers and dimersat the N-terminus, as well as preservation of secondary structure withsuccinyl-leptin.

EXAMPLE 3

This example describes the receptor binding studies performed on each ofthe leptin conjugates prepared in Example 1. Each of the leptinconjugates prepared in Example 1 was evaluated using an in vitroreceptor binding assay which measures the relative affinity of leptinconjugates based on their ability to displace radiolabeled human leptinfrom a human leptin receptor expressed in immobilized cell membranes. Asevidenced by the FIG. 10 data, the chemically modified isoforms,succinyl-, DTPA-, and EDTA² -leptin each showed relative affinities forhuman leptin receptor equal to the unmodified leptin over the entirerange of ligand binding (˜1-100 ng/mL), with ED₅₀ 's of approximately 10ng/mL.

The Example 3 data thus show that the monosubstituted succinyl-leptin,monosubstituted DTPA-leptin dimer, and EDTA² -leptin dimer demonstratepreservation of in vitro receptor binding activity as compared tounmodified leptin.

EXAMPLE 4

This example describes the solubility testing performed on each of theleptin conjugates prepared in Example 1. The leptin conjugates weredialyzed into PBS then concentrated with CentriPrep concentrators, 10kDa molecular weight cutoff (Amicon) to the point that precipitates wereobserved. The sample was clarified by centrifugation and the conjugateprotein concentration in the supernatant determined. The samples werethen kept at room temperature (≈22° C.) for 48 hours and at regular timepoints centrifuged and the conjugate protein concentration in thesupernatant redetermined. The solubility of the conjugate protein in PBSis thus defined as the steady state protein concentration at roomtemperature observed in the supernatant after centrifugation (see Table2).

                  TABLE 2    ______________________________________                 Maximum Solubility in PBS    Sample       (mg/ml)    ______________________________________    unmodified leptin                 3.2    succinyl-leptin                 8.4    DTPA-leptin  31.6    EDTA.sup.2 -leptin                 59.9    ______________________________________

The Table 2 data shows that the monosubstituted succinyl-leptin,monosubstituted DTPA-leptin, and monosubstituted EDTA² -leptin havesubstantially improved solubility as compared to unmodified leptin, withthe monosubstituted EDTA² -leptin showing dramatically enhancedsolubility.

EXAMPLE 5

This example describes the in vivo bioactivity studies performed on theleptin conjugates prepared in Example 1. The described leptin conjugateswere tested in both mouse and dog animal models to determine bioefficacyrelative to the unmodified leptin. Mice were injected daily for 5-7 dayswith monosubstituted succinyl-leptin, DTPA-leptin dimer, DTPA-leptinmonomer and EDTA² -leptin dimer at dosages of 1, 10 and 50 mg/kg bodyweight. Bioefficacy was measured as a percentage weight loss from day 0,normalized to the vehicle alone control and compared to the weight lossobserved with the unmodified protein. All samples for dosages of 1 and10 mg/kg were formulated in PBS at 0.2 and 2.0 mg/ml respectively.Higher dosages were formulated in PBS at 20-50 mg/ml for the chemicallymodified forms, however the solubility limits of the unmodified leptinnecessitated its formulation at high concentrations in a pH 4 acetatebuffer. In addition dogs were injected with 0.05, 0.15 and 0.5 mg/kgdaily dosages of succinyl-leptin at 5 mg/ml over 28 days whilemonitoring weight loss followed by a recovery period.

Bioactivity, as judged by drug induced weight loss in animal models, forsuccinyl leptin was equivalent to the unmodified leptin in both dogs andmice (FIG. 11). Similarly, both DTPA-leptin monomers and dimers andEDTA² -leptin dimers caused equivalent weight loss in mice as comparedto the unmodified leptin (FIGS. 11 & 12).

The FIG. 11 & 12 data show that the monosubstituted succinyl-leptin,monosubstituted DTPA-leptin monomers and dimers, and EDTA² -leptin dimerdemonstrate preservation of in vivo bioefficacy as compared tounmodified leptin.

EXAMPLE 6

This example describes the injection site evaluation performed on theleptin conjugates prepared in Example 1. Tissue sections from theinjection sites of three mice from each dosing group were examinedhistochemically. Injection site pathology's which were identified andscored were necrosis, suppurative (mixed cell infiltrate composed ofeosinophils and neutrophils), mononuclear cells (macrophages), leptinprecipitates (characterized as either fine ppt. or largedeposits/clumps) and giant cells. Each reaction was scored using thefollowing grading system:

    ______________________________________           0          Normal           0.5-1      Minimal change           1.5-2      Mild change           2.5-3      Moderate change           3.5-4      Marked change           4.5-5      Massive change    ______________________________________

The averaged sum of the scores for each animal were used to define anoverall biocompatibility score using the following scoring key:

    ______________________________________           0-2           Normal           3-5           Minimal           6-10          Mild           11-20         Moderate           21-30         Marked           >30           Severe    ______________________________________

Although high concentrations of succinyl-leptin were marginally solublein PBS at pH 7.0, for the purposes of injection site testing, samples ofsuccinyl-leptin at 20 mg/ml remained soluble in PBS at pH 7.2 and at 50mg/ml in PBS at pH 7.5. Table 3 shows the injection site evaluationcomparing unmodified leptin at 50 mg/mL delivered in pH 4.0, acetatebuffer vs. monosubstituted succinyl-leptin at 50 mg/mL in pH 7.5, PBS,after 7 days.

                                      TABLE 3    __________________________________________________________________________           Dose               Volume         Fine Large                                       Giant    Treatment           mg/kg               mL  Necr.                       Supp.                          Mono.                              Precip                                   Deposit                                       Cells    __________________________________________________________________________    Acetate Buf           0   20  0   0.5                          1   0    0   1           0   20  0   0  0.5 0    0   0           0   20  0   0.5                          1   0    0   0    Unmod. Leptin           50  20  0   3  2   1    4   1           50  20  0   2.5                          2   1    4   2.5           50  20  0   1.5                          2   0    1.5 1    PBS Buffer           0   20  0   0.5                          0.5 0    0   0           0   20  0   0.5                          0.5 0    0   0           0   20  0   0  0   0    0   0    Succ-leptin           50  20  0   1  1.5 0    0   0           50  20  0   2  1   0    0.5 0.5           50  20  0   1.5                          0.5 0    0   0    __________________________________________________________________________

As depicted in Table 3, monosubstituted succinyl-leptin, at highconcentration dosages, showed improvement in every category of injectionsite pathology relative to the unmodified leptin, with the most dramaticimprovement seen with the almost complete elimination of leptinprecipitates and giant cells in the injection sites.

Table 4 shows the injection site evaluation comparing unmodified leptinat 43 mg/mL delivered in pH 4.0, acetate buffer vs. monosubstitutedsuccinyl-leptin at 43 mg/mL in pH 4.0, acetate buffer, after 7 days.

                                      TABLE 4    __________________________________________________________________________           Dose               Volume            Biocomp.    Treatment           mg/kg               mL  Necr.                      Supp.                         Mono.                             Precip                                 Score                                      Reaction    __________________________________________________________________________    Acetate Buf.           0   20  0  1  1   0   2    normal           0   20  0  0  0   0   2    normal           0   20  0  0  0.5 0   2    normal    Unmod. leptin           43  20  2  4  3   3.5 27   marked           43  20  1  3  3   2.5 27   marked           43  20  1.5                      3.5                         2.5 3   27   marked    Succ-leptin           43  20  0.5                      2  1.5 0.5 10   mild           43  20  0.5                      1.5                         1.5 0   10   mild           43  20  0  1  1   0   10   mild    __________________________________________________________________________

The Table 4 data shows that, surprisingly, it was also observed thathigh concentrations of monosubstituted succinyl-leptin could bedelivered in pH 4, acetate buffer and still demonstrate the dramaticimprovements in injection site reactions observed when monosubstitutedsuccinyl-leptin was delivered in PBS.

Table 5 shows the injection site evaluation comparing unmodified leptinat 20 mg/mL delivered in pH 4.0, acetate buffer vs. monosubstitutedDTPA-leptin dimer at 20 mg/mL in PBS, after 7 days.

                                      TABLE 5    __________________________________________________________________________           Dose               Volume            Biocomp.    Treatment           mg/kg               mL  Necr.                      Supp.                         Mono.                             Precip                                 Score                                      Reaction    __________________________________________________________________________    Acetate Buf           0   80  0  0.25                         1   0   3    minimal           0   80  0  0  0.5 0   1    normal           0   80  0.25                      0.25                         1   0   4    minimal    Unmod. Leptin           20  80  0  2.5                         2.5 2   16   moderate           20  80  0.25                      3.5                         3   2   22   marked           20  80  0.25                      3  3   2.5 21   marked    PBS Buffer           0   80  0  0  0   0   0    normal           0   80  0  0  0.5 0   1    normal           0   80  0  0  0   0   0    normal    DTPA-lep           20  80  0.25                      1.5                         2   0   11   moderate    dimer  20  80  0  1  1.5 0   7    mild           20  80  0  1.5                         2   0   10   mild    __________________________________________________________________________

Table 6 shows the injection site evaluation comparing unmodified leptinat 20 mg/mL delivered in pH 4.0, acetate buffer vs. monosubstitutedEDTA² -leptin dimer at 20 mg/mL in PBS, after 7 days.

                                      TABLE 6    __________________________________________________________________________           Dose               Volume            Biocomp.    Treatment           mg/kg               mL  Necr.                      Supp.                         Mono.                             Precip                                 Score                                      Reaction    __________________________________________________________________________    Acetate Buf           0   100 0  0.25                         1   0   3    minimal           0   100 0  0.5                         1   0   4    minimal           0   100 0  0.5                         1   0   4    minimal    Unmod. Leptin           100 100 0  2  3   3   18   moderate           100 100 0.5                      2  3   3   18   moderate           100 100 0  2  2.5 2   14   moderate    PBS Buffer           0   100 0  0  0.5 0   1    normal           0   100 0  0.25                         0.25                             0   1    normal           0   100 0  0.25                         0.25                             0   1    normal    EDTA-lep           100 100 0  1.5                         2   0   9    mild    dimer  100 100 0  1.5                         1.5 0   8    mild           100 100 0.5                      2.5                         3   0   16   moderate    __________________________________________________________________________

As depicted in Tables 5 & 6, DTPA-leptin dimers (Table 5) or EDTA²-leptin dimers (Table 6) can be administered to mice at highconcentration in PBS demonstrating the same improvement in injectionsite pathology as observed with succinyl-leptin. These conjugateshowever, are substantially more soluble in pH 7, PBS and thus providefor a more rugged formulation in this buffer.

In sum, the Example 6 data shows that the monosubstitutedsuccinyl-leptin, monosubstituted DTPA-leptin monomers and dimers, andEDTA² -leptin monomers and dimers do not precipitate at the injectionsite when dosed at high concentrations, and importantly, demonstratesubstantial improvement in the adverse injection site reactions observedwith the unmodified leptin.

Materials and Methods

1. Preparation of recombinant human methionyl-leptin protein

The present recombinant human methionyl-leptin (rhu-met-leptin) may beprepared according to the above incorporated-by-reference PCTpublication, WO 96/05309 at pages 151-159. For the present workingexamples, a rhu-met-leptin was used which has (as compared to the aminoacid sequence at page 158) a lysine at position 35 instead of anarginine, and an isoleucine at position 74 instead of an isoleucine.Other recombinant human leptin proteins may be prepared according tomethods known generally in the art of expression of proteins usingrecombinant DNA technology.

While the present invention has been described in terms of certainpreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

What is claimed is:
 1. Substantially homogenous preparation ofmonsouccinylated leptin, wherein said monosuccinylated leptin ismodified exclusively at the N-terminus.
 2. Substantially homogenouspreparation of monosuccinylated leptin produced by a process comprisingthe steps of: (a) reacting leptin with 3-7 fold molar excess of succinicanhydride to form a reaction mixture; (b) stirring said reaction mixture2-16 hours at 4° C. while maintaining the pH at 7.0; (c) dialyzing saidreaction mixture against 20 mM Tris-HCl, pH 7.2; and (d) isolating saidmonosuccinylated leptin from said reaction mixture, wherein saidmonosuccinylated leptin is modified exclusively at the N-terminus.
 3. Apharmaceutical composition comprising substantially homogenouspreparation of monosuccinylated leptin of claim 1 or 2 and apharmaceutically acceptable carrier.